Aspirin ('acetylsalicylic acid') inhibits the formation of prostaglandins (PGs) that cause inflammation, swelling, pain and fever. Recent studies also suggest that aspirin may have other modes of action, including the induction of nitric oxide synthesis. We recently observed that aspirin is hydrolyzed to salicylic acid, a potent antioxidant and a hydroxyl radical trapping agent, by human plasma and HDL subfraction. Hydrolysis was competed for by substrates of "aryl esterases" that prompted us to propose that paraoxonase 1 (PON 1) type of enzymes might be involved in the metabolism of aspirin. PON 1 is synthesized in the liver and the enzyme protein has also been reported to retard the accumulation of lipid peroxides in low density lipoprotein (LDL) and has the ability to reduce lipid hydroperoxides to hydroxides. PON 1 activity has been reported to be decreased in cardiovascular disease (CVD), and in diabetes. Specific Aims: Coupled with the observation in literature that those who consumed aspirin or statins showed increased plasma PON 1 activity and aspirin and paraoxon induced the expression of PON 1 gene (preliminary results), we propose that that PON 1 gene might be induced by its substrates. In order to explore these observations further, we propose the following specific aims: 1. To determine whether PON 1 is involved in the hydrolysis of aspirin. Using both in vitro methodology as well as mice deficient in PON 1, we will determine whether PON 1 plays a role in the hydrolysis of aspirin in the plasma. 2. To determine whether PON 1 activity is essential for the anti-atherosclerotic activity of aspirin. Using PON 1 -/-/apo E-/- mice, we will determine whether salicylate and not aspirin has the ability to protect against atherosclerosis. To determine whether eNOS activity is essential for the anti-atherosclerotic activity of aspirin. Using eNOS/apo E-/- mice, we will determine whether PON-1 activity is increased in these animals and whether induction of PON 1 alone is sufficient to afford atherosclerotic protection in the absence of eNOS. 3. To determine whether the expression of PON 1 gene is induced by aspirin. Using Hep G2 cells and intact animals, we will determine the induction of PON 1 gene expression by common substrates of PON 1. The mechanisms involved in such induction will be determined. Implications: These studies would pave way for designing better class of PON activators that may serve as important deterrents of not only atherosclerosis but also diabetes and other diseases in which deficiencies in PON 1 have been noted. PUBLIC HEALTH RELEVANCE: Aspirin (acetylsalicylic acid) is effective against inflammation, swelling, pain and fever. Recent studies also suggest that aspirin may have other modes of actions. We observed that aspirin is hydrolyzed to salicylic acid by human plasma and HDL, presumably by paraoxonase 1 (PON 1). In this application, we propose that the actions of aspirin might be mediated by salicylate. We also explore the mechanisms by which PON 1 could be induced by aspirin.